Molecular biology techniques allow for the insertion of a gene or genes of interest into an expression vector, transformation of a cell with the expression vector, and the expression of a protein of interest in the transformed cell. The inserted genes can be from the same cell type or the same organism as the transformed cells, or can be from different organisms or different cell types, or can even be synthetic constructs. Commonly, the expression vector is an independently replicating piece of DNA such as a plasmid and contains an origin of replication, a multiple cloning site, and one or more regulatory elements. Different selective markers, such as genes for antibiotic resistance, may also be included as part of plasmids. Linear nucleic acids are subject to degradation by nucleases, and individual genes do not contain all the regulatory elements required for their own expression in cells. Thus, ligase enzymes are employed as part of the process of inserting a gene or genes of interest into a plasmid or other vector.
Traditional nucleic acid ligases catalyze the formation of a covalent bond between a 3′ hydroxyl group and a 5′ phosphate group. A variety of ligases are commercially available; some are optimized for ligation of sticky ends, while others can perform blunt end ligations. Many DNA ligases repair single strand breaks in duplex DNA, although some ligases are also able to repair double strand breaks.
However, conventional methods for ligation in molecular biology possess some significant disadvantages. Ligation with commercially-available ligases requires long periods of incubation at reduced temperatures (usually 16° C. overnight), the use of enzymes and the optimization of conditions for best enzyme performance, and high ratios of insert DNA to vector for multiple gene insertions (sometimes up to 6:1). Even with well-established protocols, many ligation reactions do not proceed to completion with high yields. Further, some ligation protocols involve the use of polyethylene glycol (PEG), which can inhibit later attempts at transformation.
Once the gene or genes of interest have been ligated into the expression vector, the vector must be inserted into the cell in which the gene(s) will be expressed; this process is known as transformation. Cells to be transformed may be naturally competent (i.e., able to take up exogenous nucleic acids) or may be made competent through various procedures. Several methods of transformation are commonly practiced. In one method of chemical transformation, competent cells and the vector to be taken up, along with excess carrier DNA, are placed in a solution containing lithium acetate and PEG. This solution is then incubated at 30° C.; dimethylsulfoxide (DMSO) is added, and the sample is heat-shocked, which creates pores in the cell membrane through which the expression vector can enter.
Electroporation, meanwhile, is more effective than chemical transformation. Electroporation can be practiced on nearly any kind of cell, including bacteria, yeast, and plant protoplasts. However, electroporation requires direct contact of an electrode with a solution containing the cells to be transformed. This is commonly accomplished in a special cuvette with aluminum sides. Electroporation creates an electromagnetic field inside a container or cuvette holding the cells to be transformed, and typically, several hundred volts are required (e.g. at room temperature, depending on cell size, from 100-500V are applied to the sample). After electroporation, cells must be handled carefully for an hour or more, or at least the time required for one cycle of cell division.
Conventional methods for transformation are inefficient, resulting in 10% or fewer cells taking up the desired exogenous DNA. Transformation efficiency can be increased through the use of time-consuming purification methods (ethanol precipitation, phenol/chloroform extraction, desalting, spin columns, gel purifications, etc.). In the case of electroporation, costly equipment and specialized sample holders are needed, making it difficult to process many samples in parallel. Further, heat shock steps may damage some cells.
Thus, a need exists for faster, more efficient methods to ligate nucleic acid fragments and to transform cells with exogenous DNA. Ideally, such a method would be substantially heat-free, would require fewer reagents and steps than conventional molecular biology techniques, and would result in improved ligation yields and transformation efficiencies. The present application addresses these needs.